Algae, Tree, Herbs, Bush, Shrub, Grasses, Vines, Fern, Moss, Spermatophyta, Bryophyta, Fern Ally, Flower, Photosynthesis, Eukaryote, Prokaryote, carbohydrate, vitamins, amino acids, botany, lipids, proteins, cell, cell wall, biotechnology, metabolities, enzymes, agriculture, horticulture, agronomy, bryology, plaleobotany, phytochemistry, enthnobotany, anatomy, ecology, plant breeding, ecology, genetics, chlorophyll, chloroplast, gymnosperms, sporophytes, spores, seed, pollination, pollen, agriculture, horticulture, taxanomy, fungi, molecular biology, biochemistry, bioinfomatics, microbiology, fertilizers, insecticides, pesticides, herbicides, plant growth regulators, medicinal plants, herbal medicines, chemistry, cytogenetics, bryology, ethnobotany, plant pathology, methodolgy, research institutes, scientific journals, companies, farmer, scientists, plant nutrition
Select Language:
 
 
 
 
Main Menu
Please click the main subject to get the list of sub-categories
 
Services offered
 
 
 
 
  Section: General Biotechnology / Plant Biotechnology
 
 
Please share with your friends:  
 
 

Biotechnological Applications of Plant Cell, Tissues and Organ Cultures

 
     
 
Transgenic Plants
The plants,in which a functional foreign gene has been incorporated by any biotechnological methods that generally not present in plant, are called transgenic plants. However, a number of transgenic plants carrying genes for traits of economic importance have either been released for commercial cultivation or are under field trials. There are several methods discussed in previous sections of biotechnology in this website which are used in gene transfer. These includes: (i) electroporation, (ii) particle bombardment, (iii) microinjection, (iv) Agrobacterium-mediated gene transfer, (v) co-cultivation (protoplast transformation) method, (vi) leaf disc transformation method, (vii) virus-mediated transformation, (viii) pollen-mediated transformation, (ix) liposome-mediated transformation, etc. Initially, some plants were produced by using reporter genes (see preceding section). Later on several genes for known traits of economic importance were incorporated into many crop plants. In others promoter sequences have been used that reduced/enhamced tissue specific expression of the adjacent genes according to requirement In some cases antisense RNA genes have been introduced to inhibit expression of some existing genes in a desirable manner. All these approaches led to the development of transgenic crop plants of economic importance. More than 1000 field trial tests with transgenic crop plant have been conducted. Some of the commercially grown transgenic crop plants in developed countries are: 'Flavr Savri' and 'Endless Summer' tomatoes, 'Freedom IF squash, 'High-lauric' rapeseed (canola) and 'Roundup Ready' soybean, etc. During 1995, in the USA full registration was granted to genetically engineered Bt gene containing insect resistant 'New Leaf (potato), 'Maximized (corn), 'BollGard' (cotton) (Gupta, 1996). 

So far more than 60 transgenic dicot plants including herbs, shrubs and trees and several monocots (e.g. maize, oat, rice, wheat, etc.) have been produced. In future the number of these crops certainly will go up. These transgenic plants contain certain selected traits such as herbicide resistance, insect resistance, virus resistance, seed storage protein, modified ripening, modified seed oil, agglutinin, etc. (Table 9.5). Moreover, in the light of future need the transgenic plants are being looked up as bioreactor for molecular farming i.e. for the production of novel biomedical drugs such as growth hormones, vaccines, antibodies, interferon, etc. (see preceding section).
 

Content

Applications in agricultures

 

Improvement of hybrids

 

Production of encapsulated seeds

 

Production of disease resistant plants

 

Production of stress resistant plants

 

Transfer of nif genes to eukaryotes

 

Future prospects

Applications in horticulture and forestry

 

Micropropagation

 

In Vitro Establishment of Mycorrhiza

Applications in Industry

 

Products (Secondary metabolites) from Cell Culture

 

 

Cell suspension and biotransformation

 

 

Factors affecting product yield

 

Secondary Metabolites from Immobilized Plant Cells

 

Future of Plant Tissue Culture Industry in India

Transgenic plants

 

Selectable markers and their use in transformed plants (cat gene, nptll gene, lux gene, lacZ gene)

 

Transgenic plants for crop improvement

 

 

Insect resistant transgenic plants

 

 

Herbicide resistant transgenic plants

 

Molecular farming from transgenic plants

 

 

Immunotherapeutic drugs (edible vaccines, edible antibodies, edible interferon)


Table 9.5. Transgenic plants produced by different methods.

 

Plants groups

Transgenic plant species

1. Gymnosperms Picea glauca

 

 

 

2.

Angiosperms

 

 

(i) Monocots

Asparagus sp., Dactylis glomerata, Secale, cereale, Oryza sativa, Triticum aestivum, Zea mays, Avena sativa, Festulaca arundinacea.

 

 

 

 

(ii) Dicots

Armorcia sp., Nicotina tabaccum, N. plumbaginifolia, Petunia hybrida, Lycopersicon esculentum, Solanum tuberosum, S. melangana, Arabdiopsis thaliana, Lactuca sativa, Apium grareolens, Helianthus annus, Linum usitatisimum, Brassica napus, B. oleracea, B. oleracea var capita, B. compestris, Gosssypium hirsutum, Beta vulgaris, Glycine max, Pisum sativum, Medicago sativa, M. varia, Lotus corniculanum, Vigna aconitifolia, Cuccumis sativus, C. melo, Cichorium intybus, Daccus carrota, Glycorrhiza glabra, Digitalis purpurea, Ipomoea batata, I. purpurea, Fragaria sp., Actinidia sp., Carica papaya, Vitis vinifera, Dianthus caryophyllus, Vaccinium macrocarpon, Chrysanthemum sp., Rosa sp., Populous sp. Melus sylvestris, Pyrus communis, Azadirachta indica, Juglans regia.

Selectable Marker Genes and Their Use in Transformed Plants
When plant cells are transformed by any of the transformation methods as given earlier, it is necessary to isolate the transformed cells/tissue. However, it is possible to do now. There are certain selectable marker genes present in vectors that facilitate the selection process. In transformed cells the selectable marker genes are introduced through vector. The transformed cells are cultured on medium containing high amount of toxic level of substrates such as antibiotic, herbicides, etc. For each marker gene there is one substrate (Table 9.6). For a model transgenic system, tobacco is the most common plant that is found everywhere. The young explants such as leaf discs are aseptically cut into pieces. These pieces are transferred onto tissue regeneration medium supplemented with an antibiotic, kanamycin. From the transformed discs shoots grow directly. The cells which do not undergo transformation will die due to kanamycin. Therefore, antibiotics and herbicides should be used carefully because even in low concentration many cells are damaged. When regeneration has accomplished, selection should be done thereafter. Besides, another difficulty associated with successful selection is the regeneration of shoots from transformed calli because the explants may be heterogeneous and non-transformed cells could not be selected. Therefore, such methods should be used that can ensure escape of only few non-transformed shoots from selection. However, it is ensured by using leaf discs as only the cells which are in direct contact of medium containing antibiotic/herbicide will undergo regeneration. Table 9.6. Selectable marker genes of vectors and their applications.

 

Substrates used for selection

Marker genes

1.

Antibiotics

 

 

Bleomycin

Gene ble (unknown enzyme)

 

G418, Kanamycin, Neomycin

Neomycin phosphotransferese (nptll)

 

Gentamycin

Gentamycin acetyl transferase (gat)

 

Hygromycin B

Hygromycin phosphotransferase (hpt)

 

Methotrexate trimethoprim

Dihydrofoate reductase (dfr)

 

Streptomycin

Streptomycin phosphotransferase (spt)

2.

Herbicides

 

 

Chlorosulfuron imidazolinones

Mutant form of acetolactase synthase (als)

 

Bromoxynil

Bromoxynil nitrilase (bnl)

 

Glyphosate

5-enolpyruvate shikimate-3 –phosphate (EPSP)-synthase (aroA)

 

PPT (L-phosphinothricin, also called bialaphos)

Phosphinothricin acetyltransferase (bar)

In addition, there is an alternative procedure where there will be no selection pressure imposed on cells/shoot that develop from explants. In this method, samples of tissue from regenerated shoot are taken, the samples are tested for expression of a marker gene. There is a number of marker genes which are commonly described as reporter genes or scoreable genes or screenable genes (Table 9.7). Some of the reporter genes which are most commonly used in plant transformation are: cat, gus, lux, nptll., etc. They are briefly discussed as below :

(i) Chloramphenicol acetyl transferase (CAT) gene (cat gene) : The cat gene is not used as a selectable but as reporter gene. It was first isolated from the bacterium E.coli but it is absent in higher plants and mammals. In transformed cells its presence can be detected by assaying the enzyme CAT on 32P-chloramphenicol mixed growth medium. Therefore, the enzyme uses acetyl Co-A-chloramphenicol-P32 as substrate and transfer acetyl CoA to chloramphenicol converting the later into acetyl chloramphenicol which is detected autoradiographically.

 Table 9.7. Examples of some of the reporter genes used as screenable markers.
Reporter genes Enzymes expressed Substrate / assay Identification
cat Chloramphenicol acetyl transferase 14C-chloramphenicol +Acetyl CoA (TLC) separation) Detection of acetyl chloramphenical by autoradiography
gus b-glucuronidase Glucuronides (PNPG, X-GLUC, REG, NAG) Detection of fluorescence, colorimetric, photometric
lacZ b-galactosidase b-galactosidase (X-gal) Colony color
lux Luc i ferase

Decan and FMNH2, ATP+O2+luciferin

Bioluminescence on exposure of X ray film
nptll Neomycin phosphotransferase Kan+32P-ATP (in situ assay) Detection of radioactivity
nos Nopaline synthase Arginine+ketoglutaric acid + NADH Electrophoresis
ocs Octopine synthase Arginine+pyruvate+NADH Electrophoresis

(ii) Neornycin phosphotransferase (NPTII) gene (nptII gene) : The nptIIgene confer resistance against kanamycin and detoxifies it by phosphorylation. It encodes enzyme NPTII. Presence of nptIIgene in transformed tissue can be detected by selecting them on kanamycin supplemented medium. Similarly, the presence of this enzyme is also detected in transgenic plants or transformed tissue. Commonly nos promoter is linked with nptIIgene so that synthesis of enzyme NPTII may be started well. However, if nptIIgene has adverse effect on expression of desirable gene, its expression can be improved by using an alternative approach.

Reiss et al. (1984) have discussed in detail the assay of enzyme NPTII. Firstly, NPTII is fractionated by using non-denaturing polyacrylamide gel electrophoresis (PAGE). In agar layer, radiolabelled 32P-ATP is used with kanamycin. The gel (that contain the enzyme NPTII) is covered with agar containing both 32P-ATP and kanamycin. The entire preparation is incubated at 35°C. As a result of phosphorylation of kanamycin 32P is incorporated into it, the presence of which is detected autoradiographically.

(iii) Luciferase gene (lux gene) : The lux gene is found in gloveworm (firefly) and bacteria that secretes the enzyme luciferase. Due to secretion of this enzymes the gloveworm becomes luminescent in dark. The lux gene has been transferred into tobacco through Ti-plasmid of Agrobacterium. Consequently lux gene containing bioluminescent tobacco plants were produced.

Similarly a green fluorescent protein (GFP) isolated from the jellyfish, Aequorea victoria are used as reporter gene or tag in a wide variety of organisms. These act as visible marker for gene expression.

(iv) The b-galactosidase gene (lacZ gene) : The lacZ gene that encodes b-galactosidase is a polylinker as it contains several restriction sites but maintains the proper reading frame. Most DNA fragments cloned into polylinker disrupt lacZ gene and abolish b-galactosidase activity. When a foreign gene fused with lacL gene is inserted into a microbial cell1, its presence and function can be detected. When the genetically engineered microbial/plant/animal cells contained a reporter gene is allowed to grow on medium containing a chemical X-gal (i.e. 5-bromo-4-chloro-3-indolyl-b-D-galacto-pyranoside), b-galactosidase hydrolyses X-gal, and releases an insoluble blue dye. The release of dye shows the presence of foreign gene. If there appears no color, it means the gene isdisrupted.

 
     
 
 
     



     
 
Copyrights 2012 © Biocyclopedia.com | Disclaimer